Recombinant Severe acute respiratory syndrome coronavirus 2 Spike glycoprotein (S) (N354D, D364Y) is produced in a mammalian cell expression system, ensuring proper protein folding and post-translational modifications. This product features a partial protein length from amino acids 319 to 541 with specific mutations at positions N354D and D364Y, and is C-terminally tagged with 10xHis for ease of purification and detection. The protein maintains a purity level greater than 90% as verified by SDS-PAGE.
The Spike glycoprotein of SARS-CoV-2 plays a crucial role in the virus's ability to infect host cells. It mediates viral entry by binding to the host cell receptor, angiotensin-converting enzyme 2 (ACE2). Given its essential function in viral pathogenesis, the Spike protein has become a key target for vaccine development and therapeutic interventions. This makes it a significant focus of research in understanding and combating COVID-19.
Potential Applications
Note: The applications listed below are based on what we know about this protein's biological functions, published research, and experience from experts in the field. However, we haven't fully tested all of these applications ourselves yet. We'd recommend running some preliminary tests first to make sure they work for your specific research goals.
The protein is expressed in a mammalian system, which is optimal for eukaryotic protein folding and native-like glycosylation—critical for the SARS-CoV-2 Spike Receptor Binding Domain (RBD, 319–541 aa) to adopt a functional conformation. The C-terminal 10xHis-tag is less likely to disrupt folding compared to N-terminal tags, and point mutations (N354D, D364Y) are minor, reducing the risk of major misfolding. However, no direct validation of folding (e.g., circular dichroism for secondary structure, thermal shift assays for stability) or bioactivity (e.g., ACE2 binding affinity) is provided. While mammalian expression strongly supports correct RBD structure, the impact of mutations on folding/activity remains untested—bioactivity is probable but not confirmed.
1. Receptor Binding Domain (RBD) Interaction Studies
This mutant RBD fragment (319–541 aa) covers the ACE2-binding region and can study interactions with human ACE2. The N354D/D364Y mutations may alter binding affinity/species—but this requires validation via SPR or BLI to quantify mutation-driven changes. The C-terminal His-tag enables purification/immobilization for binding assays, but results must be compared to wild-type RBD to define mutation effects.
2. Neutralizing Antibody Development and Screening
As the RBD is a key neutralizing antibody target, this mutant variant can antigenize antibodies. The His-tag simplifies capture in phage display/hybridoma screens—but antibody efficacy against mutations must be tested (e.g., neutralization assays with pseudoviruses). This is relevant for immune escape studies, but specificity to the mutant RBD (vs. wild-type) requires validation.
3. Structural and Biophysical Characterization
The defined mutant RBD fragment is suitable for structural studies (X-ray crystallography, cryo-EM). Mammalian glycosylation supports native-like structure—but the C-terminal His-tag may interfere with crystallization; tag removal (via protease cleavage) may be needed for high-resolution studies. Thermal shift assays can quantify mutation-driven stability changes.
4. Protein-Protein Interaction Mapping
The His-tagged mutant RBD can pull down cellular interactors via nickel matrices—but RBD primarily binds ACE2; "beyond ACE2" interactions are speculative. This is better framed as exploring additional low-affinity interactors, with results validated via co-IP/MS. Mutations may alter these minor interactions, but ACE2 binding remains the dominant function.
5. Immunogenicity and Vaccine Research Applications
This mutant RBD can immunize animals to assess mutation-driven immune responses—but cross-reactivity to wild-type RBD must be tested (e.g., ELISA with wild-type/mutant antigens). The His-tag aids quantification, but immune responses (antibody titer, neutralization) must be compared to wild-type to define mutation effects on immunogenicity.
Final Recommendation & Action Plan
This mammalian-expressed mutant RBD (319–541 aa, N354D/D364Y) has strong potential for RBD-focused applications due to its native-like folding/glycosylation, but validation is non-negotiable first, confirm RBD conformation (CD spectroscopy) and ACE2 binding (SPR); second, test antibody specificity/neutralization against the mutant vs. wild-type; third, optimize structural studies (e.g., tag removal for crystallization). For immunogenicity, include wild-type controls to assess mutation impacts. If validation passes, use the protein for its intended fragment-specific goals (mutation studies, antibody screening)—but always contextualize results with wild-type comparisons. If folding/binding fails, revisit expression conditions or use a glycosylation-optimized mammalian system (e.g., CHO-K1 with glycoengineering).
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